Temperature responsive adsorbent having a strong cation exchange group and method for producing the same

ABSTRACT

Provided is a temperature responsive adsorbent prepared by immobilizing a copolymer containing at least N-isopropylacrylamide to a base material surface. The copolymer has at least a strong cation exchange group. In addition, the copolymer contains the strong cation exchange group in an amount of 0.01 to 5 mol % relative to N-isopropylacrylamide in terms of monomer.

The present application is a divisional of U.S. application Ser. No.13/994,255, which is a National stags of International PatentApplication No. PCT/JP2011/079392 filed Dec. 19, 2011, which claimspriority to Japanese Application No. 2010-282373 filed Dec. 17, 2010.The disclosures of U.S. application Ser. No. 13/094,255 andInternational Patent Application No. PCT/JP2011/079392 are incorporatedby reference herein in their entireties.

TECHNICAL FIELD

The present invention relates to a temperature responsive adsorbent inwhich the surface density of efficient cation exchange groups can bechanged by temperature and a method for producing the same as well as amethod for separating physiologically active substances serving ascomponents of biomedicines by using the same.

BACKGROUND ART

An immunoglobulin (antibody) is a physiologically active substanceresponsible for an immune response. Recently, availability ofimmunoglobulin has been increased in applications such as medicines,diagnostic agents and separation/purification materials for thecorresponding antigen protein. An antibody is taken from the blood of animmunized animal, a culture solution of a cell having antibodyproducibility or an ascitic fluid culture solution of the animal.However, such blood and a culture solution containing the antibodycontain proteins other than the antibody or intricate contaminantsderived from a raw-material solution used in the cell culture. Thus, toseparate and purify the antibody from these impurity components, acomplicated and time-consuming operation is usually required.

Liquid chromatography is important for separating and purifying anantibody. Examples of a chromatographic method for separating anantibody include gel filtration chromatography, affinity-chromatography,ion exchange chromatography and reverse phase chromatography. Anantibody is separated and purified by a combination of these methods.

The ion exchange chromatography is a method of separating a counter ionpresent in a mobile phase by reversibly adsorbing it by an ion exchangegroup, which is present on the surface of an adsorbent and serves as astationary phase. As the shape of the adsorbent, beads, flat film and afilm such as a hollow fiber, are employed. These base materials, towhich a cation exchange group or an anion exchange group is bound, arecommercially available as adsorbents. The adsorbent having a cationexchange group, which has a property of mainly adsorbing an antibody andnot adsorbing most of other contaminants, has a property of easilyconcentrating and separating an antibody.

Cation exchange groups are roughly divided into a weak cation exchangegroup such as a carboxyl group and a strong cation exchange group suchas a sulfonic acid group. An adsorbent having a weak cation exchangegroup has a drawback in that the surface charge of the adsorbent changesas the pH of a mobile phase changes, with the result that a bindingcapacity to an antibody changes. Accordingly, if an adsorbent having aweak cation exchange group is used for separation/purification of anantibody, the reproducibility of separation becomes poor and therecovery rate of the antibody may decrease. In contrast, in an adsorbenthaving a strong cation exchange group, since the surface charge of theadsorbent does not change even if the pH of a mobile phase changes, thebinding capacity to an antibody does not easily change. In industrialantibody separation/purification processes, although it is difficult tokeep the pH of a mobile phase at a constant value, reproducibility ofseparation is stringently required. For this reason, an adsorbent havinga strong cation exchange group is used.

In conventional adsorbents having an ion exchange group, aphysiologically active substance adsorbed is generally eluted byincreasing the salt concentration of a mobile phase. However, it isknown that a physiologically active substance serving as a component ofa biomedicine and the like may cause an irreversible change(denaturation) by changing a salt concentration (ion strength) of amobile phase. Extreme care must be taken to determine these elutionconditions. In addition, physiologically active substances are mostlyseparated and purified in sites (low-temperature chambers) controlled atlow temperatures; however, when a physiologically active substanceadsorbed is eluted in a mobile phase of a high salt-concentration, thereis a risk that a salt precipitated at a low temperature causes cloggingof a pipe and a column.

Then, to solve a problem of conventional adsorbents having an ionexchange group, a temperature responsive adsorbent is proposed, fromwhich a physiologically active substance adsorbed can be eluted not byincreasing the salt concentration of a mobile phase but by changing anefficient surface density of ion exchange group by temperature.

Patent Literature 1 discloses a packing material containing a chargedcopolymer, a method for producing the same and a temperature responsivechromatography using the same, in which an efficient surface chargedensity of a stationary phase can foe changed by temperature change.Patent Literature 2 discloses a temperature responsive chromatographiccarrier prepared by densely immobilizing a polymer capable of changinghydration force within a temperature range of 0 to 80° C. to a basematerial surface by an atom transfer radical polymerization method.Patent Literature 3 discloses a method for producing a temperatureresponsive chromatographic carrier comprising growing a charged polymercapable of changing hydration force within a temperature range of 0 to80° C. in accordance with a reaction of an atom transfer radical methodusing isopropyl alcohol as a solvent. Patent Literature 4 discloses amethod for producing a liquid chromatographic carrier, which is preparedby covering a solid surface with a charged polymer capable of changinghydration force within a temperature range of 0 to 80° C., and which iscapable of separating a high-molecular weight physiologically activesubstance useful in the fields of e.g., biology, medicine and pharmacyunder specific conditions including an aqueous mobile phase. Non PatentLiterature 1 discloses a temperature responsive chromatographic carrierhaving a carboxyl group and prepared by an atom transfer radicalpolymerization method and a process thereof. In the Literature, monomercompositions for use in an atom transfer radical polymerization methodare disclosed including a monomer composition optimized for lysozymeseparation.

CITATION LIST Patent Literatures

Patent Literature 1: International Publication No. WO99/061904

Patent Literature 2: Japanese Patent Laid-Open No. 2007-69193

Patent Literature 3: Japanese Patent Laid-open No. 2009-85933

Patent Literature 4: International Publication No. WO01/074482

Non Patent Literatures

Non Patent Literature 1: Polymer Preprints, Japan Vol. 58, No. 2, 3T1-13(2009)

SUMMARY OF INVENTION Technical Problem

None of the aforementioned literatures disclose a temperature responsiveadsorbent having a strong cation exchange group, of which surface isgrafted with monomer compositions most suitable for purifying a proteinsuch as an immunoglobulin, a method for producing the adsorbent and amethod for applying the adsorbent. It is difficult to polymerizemonomers having cation exchange group since they extremely reduce thereaction rate of surface graft polymerization. Because of this, themonomer has not yet been sufficiently studied. Then, an object of thepresent invention is to provide a temperature responsive adsorbenthaving an optimal ratio of a strong cation exchange group relative toN-isopropylacrylamide for purification of a protein such as animmunoglobulin by temperature change, and provide a method for producingthe adsorbent and a method for applying the adsorbent.

Solution to Problem

The present inventors made research and development with a view ofattaining the aforementioned object from various angles. As a result,the present inventors found that if a temperature responsive adsorbentis produced by a surface graft polymerization method using a reactionsolution containing a monomer having a strong cation group such assulfonic acid group or a precursor of a strong cation exchange group ina ratio of 0.01 to 5 mol % relative to N-isopropylacrylamide, the strongcation groups are immobilized to a base material surface at a suitabledensity for purifying a protein such as an immunoglobulin by temperaturechange. The technique disclosed in the present invention cannot betotally expected from the prior art and development toward a novelseparation system of a physiologically active substance, which has neverbeen exist in the prior art, is expected. The present invention wasaccomplished based on such a finding.

Advantageous Effects of Invention

Based on the temperature responsive adsorbent and methods for producingand applying the adsorbent described in the present invention, a novelseparation system is proposed. If such a system is used, usefulphysiologically active substances such as proteins includingimmunoglobulins will be successfully separated/purified on an industrialscale by changing temperature.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a table summarizing experimental results ofadsorption/elution tests of an antibody by the adsorbents prepared inExamples 1 to 6 and Comparative Examples 1 and 2.

FIG. 2 shows a table summarizing experimental results of anadsorption/elution test of an antibody by the adsorbent prepared inExample 7.

DESCRIPTION OF EMBODIMENTS

Preferred embodiment of the present invention (hereinafter, referred toas “the embodiment”) will be described below in more detail. Thetemperature responsive adsorbent according to the embodiment is atemperature responsive adsorbent in which a copolymer containing atleast N-isopropylacrylamide is immobilized to a base material surface.The copolymer has at least a strong cation exchange group. Furthermore,the copolymer contains the strong cation exchange group in an amount of0.01 to 5 mol % in terms of monomer relative to N-isopropylacrylamide.The copolymer of the temperature responsive adsorbent according to theembodiment is, for example, formed by polymerizing monomer compositions,which contain monomer having the strong cation exchange group and/orprecursor monomer for introducing the strong cation exchange group in aratio of 0.01 to 5 mol % relative to N-isopropylacrylamide, by a surfacegraft polymerization method.

Examples of the shape of the base material to be used in the embodimentinclude, but are not particularly limited to, bead shape, flat-plateshape and tubular shape. In the case of the bead shape, beads havingvarious particle sizes are available. Although it is not particularlylimited, the particle size is satisfactorily 1 to 300 μm, preferably 10to 200 μm and further preferably 20 to 150 μm. If the particle size is 1μm or less, compaction of beads tends to occur in a column, with theresult that treatment of the beads at a high flow rate tends to bedifficult. In contrast, if the particle size is 300 μm or more, thespace between beads increases, with the result that some of biopolymerstend to slip out in adsorbing them.

The base material to be used in the embodiment has, for example, aplurality of pores. Although it is not particularly limited, the porediameter is satisfactorily 5 to 1000 nm, preferably 10 to 700 nm andfurther preferably 20 to 500 nm. If the pore diameter is 5 nm or less,the molecular weight of biopolymer to be separated tends to reduce. Incontrast, if the pore diameter is 1000 nm or more, the surface area ofthe base material decreases, with the result that the binding capacityto a biopolymer tends to reduce.

In the embodiment, a temperature responsive polymer having the strongcation exchange group is immobilized to the above base material.Examples of the immobilization method include, but are not particularlylimited to, an “atom transfer radical method” in which an atom transferradical polymerization initiator is immobilized to a base materialsurface and a temperature responsive polymer is allowed to grow from theinitiator through a reaction in the presence of a catalyst; and a“radiation graft polymerization method” in which a base material isirradiated with a radioactive ray to produce a radical and a temperatureresponsive polymer is allowed to grow through a reaction with theradical thus generated used as a starting point. Alternatively, as theimmobilization method, a surface living radical polymerization method,i.e., “atom transfer radical polymerization method” is known. The “atomtransfer radical polymerization method” can be suitably used since apolymer can be densely immobilized to a base material surface.

When the temperature responsive polymer is immobilized by the “atomtransfer radical polymerization method”, examples of the initiator to beused therein include, but are not particularly limited to, in the casewhere the base material has a hydroxy group as is in the embodiment,1-trichlorosilyl-2-(m, p-chloromethylphenyl)ethane,2-(4-chlorosulfonylphenyl)ethyl trimethoxysilane,(3-(2-bromoisobutyryl)propyl)dimethyl ethoxysilane and 2-bromoisobutyrylbromide. In the embodiment, a polymer chain grows from the initiator.Examples of a catalyst used therein include, but are not particularlylimited to, copper halide (Cu^(I)X) such as Cu^(I)Cl and Cu^(I)Br.Furthermore, examples of the ligand complex to the copper halideinclude, but are not particularly limited to,tris(2-(dimethylamino)ethyl)amine (Me₆TREN),N,N,N″,N″-pentamethyldiethylene triamine (PMDETA),1,1,4,7,10,10-hexamethyltriethylene tetraamine (HMTETA),1,4,8,11-tetramethyl 1,4,8,11-azacyclotetradecane (Me₄Cyclam) andbipyridine.

When the temperature responsive polymer is immobilized by the “radiationgraft polymerization method”, any means can be employed for generatingradicals from the base material; however, to uniformly generate radicalsfrom the whole base material, irradiation of ionizing radiation ispreferable. Examples of the ionizing radiation include a γ ray, anelectron beam, a β ray and a neutron beam. For irradiation of ionizingradiation on an industrial scale, an electron beam or a γ ray ispreferable. Ionizing radiation can be obtained from a radioactiveisotope such as cobalt 60, strontium 90 and cesium 137 or from an X-rayimager, an electron beam accelerator and a UV ray irradiation apparatusand others.

The irradiation dose of ionizing radiation is preferably 1 kGy or moreand 1000 kGy or less, more preferably 2 kGy or more and 500 kGy or lessand further preferably 5 kGy or more and 200 kGy or less. If theirradiation dose is less than 1 kGy, it tends to be difficult togenerate radicals uniformly. In contrast, if the irradiation doseexceeds 1000 kGy, the physical strength of the base material tends todecrease.

Graft polymerization methods using irradiation of ionizing radiation aregenerally classified roughly into a pre-irradiation method, in whichradicals are generated from a base material and then allowed to be incontact with a reactive compound, and a simultaneous irradiation method,in which radicals are generated from a base material while a film isallowed to be in contact with a reactive compound. In the embodiment,either method can be applied; however, a pre-irradiation methodproducing a small amount of oligomers is preferable.

In the embodiment, the solvent to be used in polymerization is notparticularly limited as long as it can homogenously dissolve a reactivecompound. As such a solvent, an alcohol such as ethanol, isopropanol andt-butyl alcohol; an ether such as diethyl ether and tetrahydrofuran, aketone such as acetone and 2-butanone, water or a mixture of these ismentioned.

In the embodiment, the polymer to be used for covering the base materialsurface has N-isopropylacrylamide. Poly(N-isopropylacrylamide) is knownto have a lower-limit critical temperature at 32° C. The carrierintroduced into the surface of the polymer greatly changes surfacephysical properties such as hydrophilicity/hydrophobicity at a criticaltemperature. Therefore, if this is grafted or applied as a coating tothe surface of a packing agent for chromatography, sample retentivitycan be obtained depending upon the temperature. As a result, retentionbehavior can be controlled by temperature without changing thecomposition of an eluate. Control of the lower-limit criticaltemperature to be 32° C. or more can be made by copolymerizing amonomer, which is more hydrophilic than isopropylacrylamide, such asacrylamide, methacrylic acid, acrylic acid, dimethyl acrylamide andvinyl pyrrolidone, as a hydrophilic co-monomer, withN-isopropylacrylamide. Furthermore, control of the lower-limit criticaltemperature to be 32° C. or less can be made by copolymerizing ahydrophobic monomer such as styrene, an alkyl methacrylate and an alkylacrylate, as a hydrophobic co-monomer, with N-isopropylacrylamide.

In the embodiment, the polymer for covering the base material surfacehas a strong cation exchange group such as a sulfonic acid group. As amethod for providing a strong cation exchange group is not particularlylimited. As a first method, there is a method in which acopolymerization is carried out such that a monomer having a strongcation exchange group is included to a copolymer when a temperatureresponsive polymer chain for covering a carrier surface is synthesized.Examples of a monomer unit having a sulfonic acid group include polymercomponent units having sulfonic acid such as (meth)acrylamide alkylsulfonic acid, vinyl sulfonic acid, acrylamide t-butyl sulfonic acid andstyrene sulfonic acid.

For example, in the case where at least a portion of monomer units of acopolymer is a moiety derived from a vinyl monomer having a sulfonicacid group, such as vinyl sulfonic acid, the sulfonic acid group bindsto the main chain without the aid of a linker. For this, no hydrophobicinteraction occurs between the linker and an antibody. As a result, whenan antibody is eluted from the base material surface by temperaturechange, the elution amount by temperature change can be increased. Notethat at least a portion of the monomer units of a copolymer having astrong cation exchange group can be represented by the followingchemical formula (1):—CR₁R₂—CR₃(—SO₃H)—  (1)where R₁, R₂, R₃ are each independently H or Me.

In the embodiment, as a second method for providing a strong cationexchange group to the polymer for covering the base material surface,there is a method comprising performing copolymerization of a monomerincluding a monomer having a “precursor for introducing a strong cationexchange group” and thereafter converting the precursor into thesulfonic acid group. Note that, the “precursor for introducing a strongcation exchange group” can include a “precursor of a strong cationexchange group”. The “precursor of a strong cation exchange group”refers to, for example, a strong cation exchange group provided with aprotecting group. As a monomer having a sulfonic acid group precursor,phenyl vinyl sulfonate and the like are mentioned but is not limited tothese in the embodiment.

In the embodiment, as a third method for providing the strong cationexchange group to the polymer for covering the base material surface,there is a method comprising copolymerizing a monomer including amonomer having a functional group capable of providing a strong cationexchange group as a precursor monomer for introducing the strong cationexchange group, and thereafter converting the functional group capableof providing a strong cation exchange group into the sulfonic acidgroup. Examples of the monomer having a functional group capable ofproviding a strong cation exchange group include styrene and glycidylmethacrylate. In the case where a monomer having a strong cationexchange group is polymerized in accordance with a surface livingradical polymerization method, a sufficient polymerization rate is notoften obtained; however, if a precursor monomer for introducing a strongcation exchange group at least a part of which is a methacrylic acidderivative or an acrylic acid derivative such as glycidyl methacrylateis used, a sufficient polymerization rate can be obtained.

Furthermore, by virtue of the presence of a methacrylic acid derivativeor an acrylic acid derivative as at least a portion of the monomer unitsof the copolymer having the strong cation exchange group, hydrophobicinteraction between the base material or the other part of the copolymerand an antibody can be suppressed, increasing the elution amount ofantibody in eluting the antibody from the base material surface bytemperature change.

Furthermore, by virtue of the presence of a methacrylic acid derivativeor an acrylic acid derivative as at least a portion of the monomer unitsof the copolymer having the strong cation exchange group, at least aportion of the monomer units of the copolymer having the strong cationexchange group has a group represented by the following chemical formula(2) or (3).—CH(—OH)—CH₂—SO₃H  (2)—CH(—SO₃H)—CH₂—OH  (3)

The sulfonic acid group of the monomer unit represented by the abovechemical formula (2) binds to the main chain via a linker at leastcontaining —CH(—OH)—CH₂—. Furthermore, the sulfonic acid group of themonomer unit represented by the above chemical formula (3) binds to themain chain via a linker at least containing —CH—. Since steric hindranceis suppressed by the linker, an antibody can quickly bind to thesulfonic acid group. Furthermore, the monomer units represented by theabove chemical formulas (2) and (3) each have a hydroxy group in theproximity of a sulfonic acid group. By virtue of this, hydrophobicinteraction between the base material or the other part of the copolymerand an antibody can be suppressed by the hydroxy group, increasing theelution amount of antibody in eluting the antibody from the basematerial surface by temperature change.

In the embodiment, the monomer composition having the monomer having thestrong cation exchange group and/or the precursor monomer forintroducing the strong cation exchange group in a ratio of 0.01 to 5 mol% relative to N-isopropylacrylamide is subjected to polymerization inaccordance with the surface graft polymerization method. The above ratiois preferably 0.1 to 4 mol %, more preferably 0.2 to 3 mol %, furtherpreferably 0.3 to 2 mol % and most preferably 0.5 to 1.5 mol %. If theabove ratio exceeds 5 mol %, the amount of strong cation exchange grouprelative to the amount of N-isopropylacrylamide in the copolymer becomesexcessive. As a result, the adsorption amount of immunoglobulin to thetemperature responsive adsorbent increases; however most of theimmunoglobulin adsorbed tends to be unsuccessfully eluted by temperaturechange. In contrast, if the above ratio is less than 0.01 mol %, theamount of strong cation exchange group to be introduced is excessivelylow. As a result, the adsorption amount of immunoglobulin itself tendsto decrease.

In the embodiment, a copolymerization ratio (composition) of the monomerunit having the strong cation exchange group relative toN-isopropylacrylamide can be quantitatively determined by analyzing thecopolymer immobilized to the base material surface. In analyzing thecopolymerization ratio, various analysis approaches such as elementanalysis and NMR can be employed. Analyzing the copolymerization ratioafter the copolymer is isolated from the base material is preferable inview of analysis accuracy since the effect of the base material uponanalysis can be eliminated. If the copolymer cannot be isolated from thebase material, the copolymer may be polymerized in a solution withoutusing the base material. In this manner, the copolymer to be used foranalysis of the copolymerization ratio can be obtained.

In the embodiment, the polymer covering the base material surface causeshydration and dehydration by changing temperature. The range of thetemperature is 0° C. to 80° C., preferably, 5° C. to 50° C. and furtherpreferably 10° C. to 45° C. If the temperature exceeds 80° C., e.g.,vaporization occurs since the mobile phase consists of water,workability tends to decrease. In contrast, if the temperature is lowerthan 0° C., the mobile phase tends to freeze.

The temperature responsive adsorbent obtained by the embodiment isusually loaded to a liquid chromatographic apparatus and used as aliquid chromatography system. Although a method of applying temperatureto a temperature responsive adsorbent in this case is not particularlylimited, for example, loading the temperature responsive adsorbent in analuminum block, a water bath, air layer or jacket adjusted to apredetermined temperature can be mentioned.

Although a separation method using the temperature responsive adsorbentaccording to the embodiment is not particularly limited; a method inwhich a desired biopolymer is once adsorbed to the temperatureresponsive liquid chromatographic carrier obtained and thereafter, thebiopolymer adsorbed is released by changing temperature to therebychange characteristics of the carrier surface, in short, a method usinga catch and release approach, is mentioned. The amount of solute to beadsorbed in this case may beyond or below the amount that the carriercan adsorb. Either method is a purification method of a solute by onceadsorbing it and thereafter releasing it by changing temperature tothereby change the characteristics of the carrier surface.

Examples of other separation methods include, but are not particularlylimited to, a method in which the temperature at which thecharacteristics of the carrier surface are changed is previously checkedand impurities are separated by changing temperature with thepredetermined temperature sandwiched in the middle. In this case, thecharacteristics of the carrier surface are greatly changed just bytemperature change, and thus, the time until a signal appears (retentiontime) is expected to greatly differ depending upon the solute. In thecase of the embodiment, the method is most effectively used if thesolute is separated by changing temperature with the predeterminedtemperature, at which the characteristics of the carrier surface aregreatly changed, sandwiched in the middle.

In the chromatography described in the embodiment, a buffer may be usedas a mobile phase and no organic solvent is required. The buffer hereinrefers to an aqueous solution containing inorganic salts. Specificexamples of the buffers include a phosphate buffer, a tris buffer and anacetic acid buffer. The buffer is not particularly limited as long as itis conventionally used. The concentration of the inorganic salts issatisfactorily 1 to 50 mmol/L, preferably 3 to 40 mmol/L and furtherpreferably 5 to 30 mmol/L. If the concentration of inorganic salts in amobile phase is lower than 1 mmol/L, the activity of a physiologicallyactive substance serving as the solute tends to be inhibited. Inaddition, the dissociation degree of the ion exchange group on thetemperature responsive adsorbent surface increases and the solute istightly adsorbed to the temperature responsive adsorbent surface, withthe result that removing the solute from the carrier surface by asubsequent operation tends to be difficult. Conversely, if theconcentration of inorganic salts is higher than 50 mmol/L, thedissociation degree of the ion exchange group on the temperatureresponsive adsorbent surface decreases and the solute is rarely retainedto a carrier surface. Finally, separating the solution tends to bedifficult.

A neutral buffer to be used in the embodiment satisfactorily has a pHvalue of 4.0 to 7.5, preferably, 4.5 to 7.0 and further preferably 5.0to 6.5. If the pH value of the buffer is higher than 7.5, animmunoglobulin (isoelectric point 7.5 to 10) is negatively charged andcauses electrostatic repulsion to the strong cation exchange group thatthe temperature responsive adsorbent of the embodiment has, with theresult that adsorption capacity tends to extremely decrease. Conversely,if a pH value is lower than 4.0, an immunoglobulin is denatured, withthe result that quality loss such as reduction in activity andgeneration of aggregates tends to occur. In the embodiment, the proteinis not particularly limited; however, since the embodiment is aseparation method using the carrier surface having the strong cationexchange group, a basic protein is preferable. Specific examples of thebasic protein include an immunoglobulin, lysozyme, hemoglobin β chain,catalase, annexin and ezrin. In particular, for purification ofimmunoglobulin, the separation method is preferably used.

If the temperature responsive adsorbent of the embodiment explained inthe above is used, an extremely useful physiologically active substancefor use in medicines, etc. can be separated and analyzed. In this case,separation can be made by a simple operation, i.e., just by changing thetemperature within a column. In addition, since no organic solvent isrequired for separation, the physiologically active substance can beseparated without being denatured.

Example 1

The embodiment will be more specifically described below based onexamples; however, these examples should not be construed as limitingthe embodiment.

In Example 1, a bead-shape temperature responsive adsorbent having asulfonic acid group was synthesized by the atom transfer radicalpolymerization method.

1) Immobilization of Initiator

Crosslinked polyvinyl alcohol beads (1 g (particle size: 100 μm)) wasmoistened with pure water and placed in a 300-mL conical flask made ofglass. To the conical flask, 200 mL of tetrahydrofuran (containing nostabilizer, manufactured by Kanto Chemical Co., Inc.), 1.23 mL of2-bromoisobutyryl bromide (manufactured by Tokyo Chemical Industry Co.,Ltd.) and 1.40 mL of triethylamine (manufactured by Wako Pure ChemicalIndustries Ltd.) were added and shaken at room temperature for 16 hours.After completion of the reaction, filtration was made and washing isperformed three times with 200 mL of ethanol and storage was made indehydrated isopropanol. In this manner, 2-bromoisobutyryl bromideserving as an atom transfer radical polymerization (ATRP) initiator wasintroduced into the surface of the crosslinked polyvinyl alcohol beads.

2) Surface Graft Polymerization

A monomer composition containing glycidyl methacrylate (GMA,manufactured by Tokyo Chemical Industry Co., Ltd.), which was aprecursor monomer of a sulfonic acid group, in a ratio of 1 mol %relative to N-isopropylacrylamide was prepared. More specifically, 18.40g of isopropylacrylamide (IPAAm, manufactured by Wako Pure ChemicalIndustries Ltd.), 0.231 g of GMA, 1.217 g of butyl methacrylate (BMA,manufactured by Tokyo Chemical Industry Co., Ltd.), 0.085 g of copper(I) chloride (CuCl, manufactured by Wako Pure Chemical Industries Ltd.),and 0.012 g of copper (II) chloride (CuCl₂, manufactured by Wako PureChemical Industries Ltd.) were dissolved in a 90 vol % aqueousisopropanol (IPA) solution (42.8 mL) and bubbled with nitrogen for 30minutes. Thereafter, to the solution, 0.221 g of tris(2-dimethylaminoethyl)amine (Me₆TREN) (manufactured by Alfa Aesar) wasadded under a nitrogen atmosphere and the mixture was stirred for 5minutes to form a catalyst of CuCl/CuCl₂/Me₆TREN. The reaction solutionwas reacted with an initiator-introduced crosslinked polyvinyl alcoholbeads under a nitrogen atmosphere and subjected to ATRP at roomtemperature for 16 hours. After completion of the reaction, the monomer,polymer and copper catalyst were washed successively with ethanol, a 50mmol/L-EDTA aqueous solution and pure water.

3) Introduction of a Sulfonic Acid Group

The graft-chain introduced beads by the atom transfer radicalpolymerization method were added to an aqueous solution (200 g) of amixture of sodium sulfite and IPA (sodium sulfite/IPA/purewater=10/15/75 wt %) and reacted at 80° C. for 24 hours to convert anepoxy group in the graft chain into a sulfonic acid group. Aftercompletion of the reaction, the beads were washed with pure water.Thereafter, the beads were added to 0.5 mol/L sulfuric acid and reactedat 80° C. for 2 hours to convert the remaining epoxy group in the graftchain into a diol group. After completion of the reaction, the beadswere washed with pure water.

4) Measurement of Copolymerization Ratio

Using a monomer composition containing glycidyl methacrylate (GMA,manufactured by Tokyo Chemical Industry Co., Ltd.), which was aprecursor monomer of a sulfonic acid group, in a ratio of 1 mol %relative to N-isopropylacrylamide, a copolymer was polymerized withoutusing a base material. More specifically, the reaction solutiondescribed in the above 2) was reacted with ethyl 2-bromoisobutyrateunder a nitrogen atmosphere and subjected to ATRP at room temperaturefor 16 hours. After completion of the reaction, the reaction solutionwas poured in dialysis membrane (Spectra/por Dialysis Membrane,MWCO1000, manufactured by Spectrum Laboratories) and successively soakedin ethanol, a 50 mmol/L-EDTA aqueous solution and pure water to removethe monomer and the copper catalyst. Subsequently, the reaction solutionwas lyophilized. The resultant copolymer was added to an aqueoussolution (200 g) of a mixture of sodium sulfite and IPA (sodiumsulfite/IPA/pure water=10/15/75 wt %) and reacted at 80° C. for 24 hoursto convert an epoxy group in the graft chain into a sulfonic acid group.After completion of the reaction, the reaction solution was poured indialysis membrane and soaked in pure water to remove sodium sulfite andIPA. Subsequently, the reaction solution was lyophilized to obtain acopolymer.

The above copolymer (30 mg) was dissolved in heavy water (670 mg) and1H-NMR was measured by a nuclear magnetic resonance apparatus (BrukerAvenve-600). Thereafter, based on the integrated value of aN-isopropylacrylamide unit-derived signal and the integrated value of asulfonic acid group-derived signal, the copolymerization ratio(composition) of a monomer unit having a strong cation exchange grouprelative to N-isopropylacrylamide was calculated. As a result, thecopolymerization ratio (composition) of the monomer unit having a strongcation exchange group relative to N-isopropylacrylamide was 0.72 mol %.

5) Measurement of Adsorption/Elution Amount of Immunoglobulin

A vacant column (Tricorn 5/20 column, manufactured by GS HealthcareJapan) was filled with the beads. The adsorption/elution test of animmunoglobulin (Venogloblin-IH blood donation, manufactured by BenesisCorporation) was performed by using a chromatography system (AKTA FPLC,manufactured by GE Healthcare Japan) by changing temperature. Anoperation for changing the temperature of the column filled with thebeads was performed by temporarily stopping the pump of thechromatography system, soaking the column in a constant-temperaturewater vessel and thereafter storing it in a warm place for 10 minutes ormore and driving the pump of the chromatography system, again.Adsorption and elution of an immunoglobulin were performed in thefollowing conditions.

(Adsorption Step)

-   -   An immunoglobulin concentration: 2.5 mg/mL    -   Adsorption buffer: 15 mmol/L acetic acid buffer (pH 6.0)    -   An immunoglobulin solution loading amount: 20 mL    -   Flow rate: 0.4 mL/min    -   Column volume: 0.54 ml    -   Adsorption temperature: 40° C.        (Washing Step)    -   Wash buffer: 15 mmol/L acetic acid buffer (pH 6.0)    -   Flow rate: 0.4 mL/min    -   Wash temperature: 40° C.        (Elution Step by Temperature Change)    -   Elution buffer: 15 mmol/L acetic acid buffer (pH 6.0)    -   Flow rate: 0.4 mL/min    -   Flow amount: 20 mL    -   Elution temperature: 2° C.        (Salt Elution Step)    -   Elution buffer: 1 mol/L acetic acid buffer (pH 6.0)    -   Flow rate: 0.4 mL/min    -   Flow amount: 20 mL    -   Elution temperature: 2° C.

After elution by temperature change, an immunoglobulin that cannot becompletely eluted by temperature change was eluted with a 1 mol/L aceticacid buffer (pH 6.0). UV absorption (280 nm) in each step was measuredand an immunoglobulin concentration was calculated in accordance withthe following expression to obtain the elution amount of immunoglobulinby temperature change.Immunoglobulin concentration (mg/mL)=absorbance at 280 nm/14×10Elution amount by temperature change (mg/mL)=immunoglobulinconcentration of fraction eluted by temperature change×liquid amount offraction eluted by temperature change/column volume(Results)

As shown in FIG. 1, the elution amount of immunoglobulin by temperaturechange is 30.7 mg/mL, demonstrating that the immunoglobulin can beeluted by temperature change. After elution by temperature change, theimmunoglobulin left on the beads was eluted by a salt buffer. As aresult, the elution amount by the salt buffer was as low as 1.4 mg/mL.From the above results, it was demonstrated that the temperatureresponsive adsorbent can be used for industrial immunoglobulinpurification.

Example 2

In a surface graft polymerization reaction, a monomer compositioncontaining glycidyl methacrylate, which was a precursor monomer of asulfonic acid group, in a ratio of 0.5 mol % relative toN-isopropylacrylamide was prepared and put in use. More specifically, atemperature responsive adsorbent was synthesized in the same manner asin Example 1 except that a reaction solution prepared by dissolvingN-isopropylacrylamide (18.40 g), glycidyl methacrylate (0.116 g), butylmethacrylate (1.217 g), copper I chloride (0.085 g) and copper (II)chloride (0.012 g) in a 90 vol % aqueous isopropanol (IPA) solution(42.8 mL) was used, and an adsorption and elution test of animmunoglobulin was performed in the same manner as in Example 1.Furthermore, a copolymerization ratio was determined in the same manneras in Example 1 except that the reaction solution having the abovecomposition was used. As a result, the copolymerization ratio(composition) of the monomer unit having the strong cation exchangegroup relative to N-isopropylacrylamide was 0.36 mol %.

(Results)

As shown in FIG. 1, the elusion amount of immunoglobulin by temperaturechange was 7.7 mg/mL, demonstrating that the immunoglobulin can beeluted by temperature change. After elution by temperature change, theimmunoglobulin left on the beads was eluted by a salt buffer. As aresult, the elution amount by the salt buffer was as low as 1.0 mg/mL.From the above results, it was demonstrated that the temperatureresponsive adsorbent can be used for industrial immunoglobulinpurification.

Example 3

In a surface graft polymerization reaction, a monomer compositioncontaining glycidyl methacrylate, which was a precursor monomer of asulfonic acid group, in a ratio of 2 mol % relative toN-isopropylacrylamide was prepared and put in use. More specifically, atemperature responsive adsorbent was synthesized in the same manner asin Example 1 except that a reaction solution prepared by dissolvingN-isopropylacrylamide (18.40 g), glycidyl methacrylate (0.462 g)butylmethacrylate (1.217 g), copper I chloride (0.085 g) and copper (II)chloride (0.012 g) in a 90 vol % aqueous isopropanol (IPA) solution(42.8 mL) was used, and an adsorption and elution test of animmunoglobulin was performed in the same manner as in Example 1.Furthermore, a copolymerization ratio was determined in the same manneras in Example 1 except that the reaction solution having the abovecomposition was used. As a result, the copolymerization ratio(composition) of the monomer unit having the strong cation exchangegroup relative to N-isopropylacrylamide was 1.44 mol %.

(Results)

As shown in FIG. 1, the elusion amount of immunoglobulin by temperaturechange was 21.3 mg/mL, demonstrating that the immunoglobulin can beeluted by temperature change. After elution by temperature change, theimmunoglobulin left on the beads was eluted by a salt buffer. As aresult, the elution amount by the salt buffer was as low as 7.7 mg/mL.From the above results, it was demonstrated that the temperatureresponsive adsorbent can be used for industrial immunoglobulinpurification.

Example 4

In a surface graft polymerization reaction, a monomer compositioncontaining glycidyl methacrylate, which was a precursor monomer of asulfonic acid group, in a ratio of 3 mol % relative toN-isopropylacrylamide was prepared and put in use. More specifically, atemperature responsive adsorbent was synthesized in the same manner asin Example 1 except that a reaction solution prepared by dissolvingN-isopropylacrylamide (18.40 g), glycidyl methacrylate (0.694 g), butylmethacrylate (1.21 g), copper I chloride (0.085 g) and copper (II)chloride (0.012 g) in a 90 vol % aqueous isopropanol (IPA) solution(42.8 mL) was used, and an adsorption and elution test of animmunoglobulin was performed in the same manner as in Example 1.Furthermore, a copolymerization ratio was determined in the same manneras in Example 1 except that the reaction solution having the abovecomposition was used. As a result, the copolymerization ratio(composition) of the monomer unit having the strong cation exchangegroup relative to N-isopropylacrylamide was 2.16 mol %.

(Results)

As shown in FIG. 1, the elusion amount of immunoglobulin by temperaturechange was 17.1 mg/mL, demonstrating that the immunoglobulin can beeluted by temperature change. After elution by temperature change, theimmunoglobulin left on the beads was eluted by a salt buffer. As aresult, the elution amount by the salt buffer was 33.9 mg/mL. From theabove results, it was demonstrated that the temperature responsiveadsorbent can be used for industrial immunoglobulin purification.

Example 5

In a surface graft polymerization reaction, a monomer compositioncontaining glycidyl methacrylate, which was a precursor monomer of asulfonic acid group, in a ratio of 4 mol % relative toN-isopropylacrylamide was prepared and put in use. More specifically, atemperature responsive adsorbent was synthesized in the same manner asin Example 1 except that a reaction solution prepared by dissolvingN-isopropylacrylamide (18.40 g), glycidyl methacrylate (0.924 g), butylmethacrylate (1.217 g), copper I chloride (0.085 g) and copper (II)chloride (0.012 g) in a 90 vol % aqueous isopropanol (IPA) solution(42.8 mL) was used, and an adsorption and elution test of animmunoglobulin was performed in the same manner as in Example 1.Furthermore, a copolymerization ratio was determined in the same manneras in Example 1 except that the reaction solution having the abovecomposition was used. As a result, the copolymerization ratio(composition) of the monomer unit having the strong cation exchangegroup relative to N-isopropylacrylamide was 2.88 mol %.

(Results)

As shown in FIG. 1, the elusion amount of immunoglobulin by temperaturechange was 13.6 mg/mL, demonstrating that the immunoglobulin can beeluted by temperature change. After elution by temperature change, theimmunoglobulin left on the beads was eluted by a salt buffer. As aresult, the elution amount by the salt buffer was 52.2 mg/mL. From theabove results, it was demonstrated that the temperature responsiveadsorbent can be used for industrial immunoglobulin purification.

Example 6

A hollow fiber shape temperature responsive adsorbent having a sulfonicacid group was synthesized in accordance with a γ ray graftpolymerization method.

1) Surface Graft Polymerization

N-isopropylacrylamide (4.66 g), glycidyl methacrylate (0.059 g) andbutyl methacrylate (0.120 g) were dissolved in a 25 vol % aqueoust-butanol solution (200 mL) and bubbled with nitrogen for 30 minutes.This was used as a reaction solution. Polyethylene hollow fiber (0.800 g(10 cm, 4 filaments), inner diameter: 2.0 mm, outer diameter: 3.0 mm,average pore diameter: 0.25 μm,) was cooled with dry ice to −60° C.under a nitrogen atmosphere, and irradiated with γ rays at 35 kGy usingCo60 as a radiation source. The hollow fiber irradiated was allowed tostand still under a reduced pressure of 13.4 Pa or less for 5 minutesand thereafter contacted with the above reaction solution (20 mL) at 40°C. and allowed to stand still for 16 hours. Thereafter, the hollow fiberwas washed with ethanol and dried in a vacuum dryer.

2) Introduction of Sulfonic Acid Group

The hollow fiber having a graft chain introduced therein by the γ raygraft polymerization method was placed in an aqueous solution (500 g) ofa mixture of sodium sulfite and IPA (sodium sulfite/IPA/purewater=10/15/75 wt %) and reacted at 80° C. for 24 hours to convert anepoxy group in the graft chain into a sulfonic acid group. Aftercompletion of the reaction, the hollow fiber was washed with pure water.Thereafter, the hollow fiber was added to 0.5 mol/L sulfuric acid andreacted at 80° C. for 2 hours to convert the remaining epoxy group inthe graft chain into a dial group. After completion of the reaction, thehollow fiber was washed with pure water, and a film was washed withethanol and dried in a vacuum drier.

3) Measurement of Adsorption/Elution Amount of Immunoglobulin

The hollow fiber (film volume: 0.5 mL) modularized was subjected to anadsorption/elution test of an immunoglobulin (Venogloblin-IH blooddonation, manufactured by Benesis Corporation) using a chromatographysystem (AKTA FPLC, manufactured by GE Healthcare Japan) by temperaturechange. An operation for changing the temperature of the hollow fibermodule was performed by temporarily stopping the pump of thechromatography system, soaking the hollow fiber module in aconstant-temperature water vessel, and thereafter storing it in a warmplace for 10 minutes or more, and driving the pump of the chromatographysystem, again. Adsorption and elution of an immunoglobulin wereperformed in the following conditions.

(Adsorption Step)

-   -   An immunoglobulin concentration: 2.5 mg/mL    -   Adsorption buffer: 15 mmol/L acetic acid buffer (pH 6.0)    -   An immunoglobulin solution loading amount: 20 mL    -   Flow rate: 0.4 mL/min    -   Column volume: 0.54 mL    -   Adsorption temperature: 40° C.        (Washing Step)    -   Wash buffer: 15 mmol/L acetic acid buffer (pH 6.0)    -   Flow rate: 0.4 mL/min        (Elution Step by Temperature Change)    -   Elution buffer: 15 mmol/L acetic acid buffer (pH 6.0)    -   Flow rate: 0.4 mL/min    -   Amount of liquid passed through: 20 mL    -   Elution temperature: 2° C.        (Salt Elution Step)    -   Elution buffer: 1 mol/L acetic acid buffer (pH 6.0)    -   Flow rate: 0.4 mL/min    -   Amount of liquid passed through: 20 mL    -   Elution temperature: 2° C.

After elution by temperature change, an immunoglobulin that cannot becompletely eluted by temperature change was eluted with a 1 mol/L aceticacid buffer (pH 6.0). UV absorption (280 nm) of fractions in each stepwas measured and an immunoglobulin concentration was calculated inaccordance with the following expression to obtain the elution amount ofimmunoglobulin by temperature change.An immunoglobulin concentration (mg/mL)=absorbance at 280 nm/14×10Elution amount by temperature change (mg/mL)=immunoglobulinconcentration of fraction eluted by temperature change×liquid amount offraction eluted by temperature change/film volume

Furthermore, a copolymerization ratio was determined in the same manneras in Example 1 except that the reaction solution having the compositionprepared above, was used. As a result, the copolymerization ratio(composition) of the monomer unit having the strong cation exchangegroup relative to N-isopropylacrylamide was 0.72 mol %.

(Results)

As shown in FIG. 1, the elusion amount of immunoglobulin by temperaturechange was 9.2 mg/mL, demonstrating that an immunoglobulin can be elutedby temperature change. After elution by temperature change, theimmunoglobulin left on the hollow fiber was eluted by a salt buffer. Asa result, the elution amount by the salt buffer was as low as 1.2 mg/mL.From the above results, it was demonstrated that the temperatureresponsive adsorbent can be used for industrial immunoglobulinpurification.

Example 7

1) Immobilization of Initiator

Crosslinked polyvinyl alcohol beads (1 g (particle size: 100 μm)) weremoistened with pure water and placed in a 300-mL conical flask made ofglass. To the conical flask, a 3% aqueous sodium hydroxide solution (150mL) and epichlorohydrin (50 g) were added and stirred at 30° C. for 2hours. After completion of the reaction, filtration was performed andwashing was made five times with pure water (200 mL). To the beads, 100mL of a 25% aqueous ammonia solution was added and stirred at 50° C. for2 hours. After completion of the reaction, filtration was performed andwashing was made five times with pure water (200 mL). Furthermore, tothe beads, 1.75 g of 4,4-azobis(4-cyanovaleric acid) (ACV), 3.00 g of1-ethoxycarbonyl-2-ethoxy-1,2dihydroquinone (EEDQ) and 80 mL ofdimethylformamide were added and stirred at 25° C. for 24 hours. Aftercompletion of the reaction, filtration was performed and washing wasmade five times with pure water (200 mL).

2) Surface Graft Polymerization

A monomer composition containing vinyl sulfonic acid (manufactured byASAHI KASEI FINECHEM CO., LTD.), which was a sulfonic acid groupcontaining monomer, in a ratio of 2 mol % relative toN-isopropylacrylamide was prepared. More specifically, 4.66 g ofH-isopropylacrylamide, 0.09 g of vinyl sulfonic acid and 0.12 g of butylmethacrylate were dissolved in t-butyl alcohol (42.8 mL) and bubbledwith nitrogen for 30 minutes. The reaction solution was reacted with acrosslinked polyvinyl alcohol beads having an initiator introducedtherein under a nitrogen atmosphere and the reaction was performed at70° C. for 16 hours. After completion of the reaction, the monomer,polymer was washed successively with ethanol and pure water.

3) Measurement of Copolymerization Ratio

Using a monomer composition containing vinyl sulfonic acid, which was asulfonic acid group containing monomer, in a ratio of 2 mol % relativeto N-isopropylacrylamide, a copolymer was polymerized without using abase material. More specifically, the reaction solution described in theabove 2) was reacted with ACV under a nitrogen atmosphere at 70° C. for16 hours. After completion of the reaction, the reaction solution waspoured in dialysis membrane (Spectra/por Dialysis Membrane, MWCO1000,manufactured by Spectrum Laboratories) and successively soaked inethanol and pure water to remove the monomer. Subsequently, the reactionsolution was lyophilized to obtain the copolymer.

The above copolymer (30 mg) was dissolved in heavy water (670 mg) and1H-NMR was measured by a nuclear magnetic resonance apparatus (BrukerAvenve-600). Thereafter, based on the integrated value of aN-isopropylacrylamide unit-derived signal and the integrated value of asulfonic acid group-derived signal, the copolymerization ratio(composition) of the monomer unit having the strong cation exchangegroup relative to N-isopropylacrylamide was calculated. As a result, thecopolymerization ratio (composition) of the monomer unit having thestrong cation exchange group relative to N-isopropylacrylamide was 0.70mol %.

(Test and Results)

An adsorption and elution test of an immunoglobulin was performed in thesame manner as in Example 1. As shown in FIG. 2, the elusion amount ofimmunoglobulin by temperature change was 30.1 mg/mL, demonstrating thatthe immunoglobulin can be eluted by temperature change. After elution bytemperature change, the immunoglobulin left on the beads was eluted by asalt buffer. As a result, the elution amount by the salt buffer was 1.1mg/mL. From the above results, it was demonstrated that the temperatureresponsive adsorbent can be used for industrial immunoglobulinpurification.

Comparative Example 1

In a surface graft polymerization reaction, a monomer compositioncontaining glycidyl methacrylate, which was a precursor monomer of asulfonic acid group, in a ratio of 0 mol % relative toN-isopropylacrylamide was prepared and put in use. More specifically, atemperature responsive adsorbent was synthesized in the same manner asin Example 1 except that a reaction solution prepared by dissolvingN-isopropylacrylamide (18.40 g), butyl methacrylate (1.217 g), copper Ichloride (0.085 g) and copper (II) chloride (0.012 g) in a 90 vol %aqueous isopropanol (IPA) solution (42.8 mL) was used, and an adsorptionand elution test of an immunoglobulin was performed in the same manneras in Example 1.

(Results)

As shown in FIG. 1, the elusion amount of immunoglobulin by temperaturechange was 0.3 mg/mL. After elution by temperature change, theimmunoglobulin left on the beads was eluted by a salt buffer. As aresult, the elution amount by the salt buffer was 0.6 mg/mL.

Comparative Example 2

In a surface graft polymerization reaction, a monomer compositioncontaining glycidyl methacrylate, which was a precursor monomer of asulfonic acid group, in a ratio of 7 mol % relative toN-isopropylacrylamide was prepared and put in use. More specifically, atemperature responsive adsorbent was synthesized in the same manner asin Example 1 except that a reaction solution prepared by dissolvingN-isopropylacrylamide (18.40 g), glycidyl methacrylate (1.618 g), butylmethacrylate (1.217 g), copper I chloride (0.085 g) and copper (II)chloride (0.012 g) in a 90 vol % aqueous isopropanol (IPA) solution(42.8 mL) was used, and an adsorption and elution test of animmunoglobulin was performed in the same manner as in Example 1.Furthermore, a copolymerization ratio was determined in the same manneras in Example 1 except that the reaction solution having the abovecomposition was used. As a result, the copolymerization ratio(composition) of the monomer unit having the strong cation exchangegroup relative to N-isopropylacrylamide was 5.04 mol %.

(Results)

As shown in FIG. 1, the elusion amount of immunoglobulin by temperaturechange was 7.0 mg/mL. After elution by temperature change, theimmunoglobulin left on the beads was eluted by a salt buffer. As aresult, the elution amount by the salt buffer was 67.0 mg/mL.

The present application was made based on Japanese Patent Application(Japanese Patent Application No. 2010-282373) filed on Dec. 17, 2010with the Japanese Patent Office and the content thereof is incorporatedby reference.

INDUSTRIAL APPLICABILITY

A novel separation system is proposed based on the temperatureresponsive adsorbent according to the embodiment, a production methodfor the adsorbent and an application method for the adsorbent. If thissystem is used, useful physiologically active compounds such asglobulins can be fractionated by temperature change on an industrialscale.

The invention claimed is:
 1. A temperature responsive adsorbent capableof separating a physiologically active substance comprising a copolymercontaining at least N-isopropylacrylamide immobilized to a supportmaterial surface, wherein at least a portion of monomer units of thecopolymer has a sulfonic acid group, and the sulfonic acid group isintroduced into the copolymer by polymerizing monomer compositions by asurface graft polymerization method to form the copolymer, each of themonomer compositions containing a monomer having the sulfonic acid groupand/or a precursor monomer for introducing the sulfonic acid group in aratio of 0.01 to 5 mol % relative to the N-isopropylacrylamide, whereinat least a portion of the monomer units of the copolymer having thesulfonic acid group is an acrylic acid derivative or a methacrylic acidderivative and has a group represented by the following chemical formula(4) or (5):—CH(—OH)—CH₂—SO₃H  (4)—CH(—SO₃H)—CH₂—OH  (5).
 2. The temperature responsive adsorbentaccording to claim 1, wherein at least a portion of the precursormonomers for introducing the sulfonic acid group is an acrylic acidderivative or a methacrylic acid derivative and has a group representedby the chemical formula (4) or (5).
 3. The temperature responsiveadsorbent according to claim 1, wherein the surface graft polymerizationmethod is a surface living radical polymerization method.
 4. Thetemperature responsive adsorbent according to claim 1, wherein thesurface graft polymerization method is a radiation radicalpolymerization method.
 5. A temperature responsive adsorbent capable ofseparating a physiologically active substance comprising a copolymercontaining at least N-isopropylacrylamide immobilized to a supportmaterial surface, wherein at least a portion of monomer units of thecopolymer has a sulfonic acid group, and the sulfonic acid group isintroduced into the copolymer by polymerizing monomer compositions by asurface graft polymerization method to form the copolymer, each of themonomer compositions containing a monomer having the sulfonic acid groupand/or a precursor monomer for introducing the sulfonic acid group in aratio of 0.01 to 5 mol % relative to the N-isopropylacrylamide, whereinat least a portion of the monomer units of the copolymer having thesulfonic acid group is derived from a vinyl monomer having a sulfonicacid group.
 6. The temperature responsive adsorbent according to claim5, wherein at least a portion of the monomers having the sulfonic acidgroup is a vinyl monomer having a sulfonic acid group.
 7. Thetemperature responsive adsorbent according to claim 5, wherein at leasta portion of the monomer units of the copolymer having the sulfonic acidgroup is represented by the following chemical formula (8):—CR₁R₂—CR₃(—SO₃H)—  (8) where R₁, R₂, and R₃ each is independently H orMe.